Maintenance With Hypomethylating Agents After Allogeneic Stem Cell Transplantation in Acute Myeloid Leukemia and Myelodysplastic Syndrome: A Systematic Review and Meta-Analysis

Introduction Hypomethylating agents (HMAs) seem to have a range of properties favorable to post-allogeneic hematopoietic stem cell transplantation (allo-SCT) maintenance in acute myeloid leukemia (AML) patients. Materials and Methods The Embase, MEDLINE, and Cochrane Central Register of Controlled Trials databases were independently searched by two investigators to identify relevant studies published inception to 18 November 2021. These trials compared HMA maintenance to observation following allo-SCT for AML or myelodysplastic syndrome. Results The meta-analysis eligibility criteria were fulfilled by 14 studies. The overall survival and relapse-free survival of the HMA maintenance group were superior to the observation group, with a pooled risk ratio (RR) of 1.38 and 1.46, respectively. Moreover, the cumulative incidence of relapse was significantly lower in those who received HMAs. The HMA group also had lower non-relapse mortality compared with the observation group. Overall, the incidences of grades III–IV acute graft-vs.-host disease (GVHD) and chronic GVHD did not differ in both groups. However, when looking specifically at those receiving decitabine maintenance, the rate of chronic GVHD seemed to be lower compared with observation alone. Conclusions The current systematic review and meta-analysis illustrated that AML and MDS patients receiving HMA maintenance after allo-SCT had better outcomes in regards to OS, RFS, NRM, CIR as well as a reduced incidence of chronic GVHD.


INTRODUCTION
Allogeneic stem cell transplantation is the mainstay treatment for AML stratified as intermediate or unfavorable risk as well as for high-risk MDS. This therapy has demonstrated superior efficacy over non-alloSCT approaches in regards to long-term clinical outcomes (1,2). Nevertheless, even after allo-SCT, 35-45% of patients suffer from disease relapse, leading to dismal outcomes (3,4).
Several strategies have been adopted to prolong diseasefree survival. Based on the time of intervention, they can be categorized into either preemptive approaches-those commenced at the time of detection of minimal residual disease (MRD)-or prophylactic approaches-those initiated in the absence of detectable leukemia. In the case of the prophylactic approaches, both cellular and pharmacological maintenance strategies have been reported, including prophylactic donor leukocyte infusion (DLI), hypomethylating agents (HMAs), histone deacetylase inhibitors, FMS-like tyrosine kinase 3 (FLT3) inhibitors, or isocitrate dehydrogenase inhibitors (5)(6)(7). Notably, HMAs have generated considerable research interest in recent years due to their favorable side effect profile.
HMAs exhibit several properties that make them suitable for post-allo-SCT maintenance. They mediate a direct anti-leukemic effect in AML and MDS, regardless of their molecular mutation profile. Moreover, their abilities to induce a CD8+ tumor-specific T cell response, together with the expansion of regulatory T cells, lead to an epigenetically enhanced Graft vs. Leukemia (GVL) effect that is not counterbalanced by an increased risk of GVHD (8)(9)(10)(11)(12). Lastly, they are safe and well-tolerated by AML patients in remission (13).
Many studies have examined the use of azacitidine and decitabine as maintenance after allo-SCT for AML and MDS. Although the majority of the studies supported consideration of HMA maintenance therapy, the remainder did not demonstrate clear benefits (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29). A recent systematic review explored the safety and efficacy of maintenance treatment following allo-SCT in AML and MDS. It demonstrated rates of 65.6 and 56.2% for the 2-year overall survival (OS) and the relapsefree survival (RFS), respectively, of HMA-treated patients. In addition, acute and chronic GVHD were found in 39.9 and 44.4%, of patients respectively. These results suggest that HMA maintenance could be employed to prolong RFS and OS (30). Nonetheless, the benefit of HMA maintenance after allo-SCT is still uncertain. This meta-analysis was performed to review all relevant studies to compare the outcomes of patients undergoing allo-SCT for AML or MDS receiving HMA maintenance therapy with observation only.

Selection Criteria and Data Extraction
The inclusion criteria were as follows: (1) studies had to be either randomized controlled trials (RCTs) or cohort studies (prospective or retrospective); (2) the patients underwent allo-SCT for AML or MDS; (3) the studies compared two patient groups: one receiving an HMA post-allo-SCT, and the other being an observational group; and (4) the studies needed to report at least one of our primary outcomes of interest (OS, RFS, grades III-IV acute GVHD, and chronic GVHD). The secondary outcomes of interest were the CIR and NRM. Study eligibility was individually assessed by three investigators (B.P., W.O, S.K.); disagreements were resolved by consensus.
Two investigators (B.P., W.O.) utilized a standardized collection form to extract the baseline characteristic data of the patients in each group, along with details of the primary and secondary outcomes of interest. The extracted data was crosschecked to confirm its accuracy.

Definitions of Outcomes
The OS rate was defined as the time between the stem cell infusion and the time of death or last follow-up, while RFS was defined as the time interval from the stem cell infusion to the date of relapse or death from any cause. All causes of death (other than death from a relapse) were used to calculate the NRM rate.

Quality Assessment
Two investigators (B.P., W.O.) assessed the quality of each study using the Jadad scale for RCTs and the Newcastle-Ottawa scale for cohort studies (31,32).

Statistical Analysis
The Mantel-Haenszel method was used to combine the effect estimates and 95% confidence intervals (CIs) of each study, and to calculate the pooled odds ratio (OR) with 95% CI (33). A random-effects model was preferred over a fixed-effects model because it was more likely that high heterogeneity would be found among the studies. Statistical heterogeneity was calculated using Cochran's Q test, estimated by the heterogeneity (I 2 ) statistic. There were four heterogeneity levels: insignificant (I 2 values of 0-25%), low (I 2 values of 26-50%), moderate (I 2 values of 51-75%), and high (I 2 values of > 75%) (34). The presence of a publication bias was visualized by a funnel plot along with Egger's regression test. Due to a lack of clinical studies, a subgroup analysis based on the types of HMAs could not be performed. All statistical analyses were performed using the Review Manager (RevMan) software (version 5.3; The Cochrane Collaboration, Oxford, UK) and "meta" package version 5.1-0. This study was registered at www.inplasy.com as #INPLASY2021110078.

Search Results
The systematic search of the Embase, MEDLINE and Cochrane Central Register of Controlled Trials databases identified 5,680 articles, from which 1,578 duplicates were removed. This resulted in 4,102 articles available for title and abstract review. Subsequently, 4,068 articles were excluded as the article type and study design did not fulfill the inclusion criteria, or there was no report on a primary outcome of interest. The remaining 34 articles underwent full-length review and 20 of those were excluded for the aforementioned reasons. Ultimately, the eligibility criteria for our meta-analysis were met by 14 studies: two RCTs, two prospective cohort study, and 10 retrospective cohort studies (14-22, 25, 27-29). Twelve of these compared azacitidine maintenance to observation, whereas two compared decitabine maintenance to observation. Figure 1 illustrates the full literature review and selection process.

Baseline Patient Characteristics
The 14 included studies were composed of 533 patients who received HMAs as maintenance, and another 784 patients who were observed post-allo-SCT. The age of the participants varied greatly (HMA group: 2 to 78 years; and observation group: 2 to 75 years). AML accounted for the largest proportion of the disease subtypes in the HMA group (84.8%), followed by MDS (14.6%) and mixed-phenotype leukemia (0.6%). These values were similar to the corresponding proportions found in the observation group (87.5, 11.2, and 1.3%, respectively). In both groups, matched unrelated donors and matched sibling donors were the most common donor sources for allo-SCT, accounting for 45.2 and 26.8%, respectively. In addition, myeloablative conditioning regimens (68.2%) were used more frequently compared to reduced-intensity conditioning regimens (31.8%). Details of the patient characteristics, such as disease status before allo-SCT, MRD status before allo-SCT cytogenetic risk, prior treatment and HMA before allo-SCT, comorbidities and performance status [Hematopoietic Cell Transplantation-Comorbidity Index (HCT-CI)], study period and quality assessment are summarized in Table 1. Details of the donor types, stem cell source, median dose of CD34+ stem cells, conditioning regimens, GVHD prophylaxis, MRD status after allo-SCT, maintenance protocols and prophylactic DLI are listed in Table 2.

Subgroup Analysis Based on Study Design
A subgroup analysis based on the study design was performed. There is a trend for a significantly lower risk of chronic GVHD in randomized studies but not in observational studies. The CIR results appear to be similar between randomized studies and observational studies. In observational studies, other parameters which included OS, RFS were significantly better in HMAs arm. However, these parameters could not be analyzed in randomized studies subgroup due to limited number of studies (Supplementary Data 4).

Sensitivity Analyses
In total, three sensitivity analyses were conducted. The first analysis was performed on studies which recruited only patients with AML. Only RFS and CIR were significantly better in HMAs arm. In addition, there was a trend of superior OS in HMAs group. Nevertheless, other parameters could not be evaluated due to limited number of studies (Supplementary Data 5). The second analysis was performed on studies with adult patients. The OS, RFS and NRM were significantly superior in HMAs arm while the other outcomes did not show significant superiority (Supplementary Data 6). The third analysis was performed on studies with low risk of bias by selecting only studies with a Newcastle-Ottawa scale of at least eight or Jadad score of at least two. Similar results were obtained compared to the main results (Supplementary Data 7).

Quality of Evidence Using Grading of Recommendations Assessment, Development, and Evaluation Approach
The quality of evidence generated by the current systematic review and meta-analysis is moderate.

DISCUSSION
An earlier systemic review that focused on the safety and efficacy of HMAs as post-allo-SCT maintenance for AML and MDS found acceptable OS and RFS rates without a heightened GVHD rate (30). Unfortunately, a detailed analysis of the clinical outcomes of the control and treatment groups was not reported. A recent meta-analysis of HMAs and FLT3 Inhibitors as maintenance treatment for AML and MDS after allo-SCT showed a high percentage of OS and RFS (35). Due to the available limited studies comparing clinical outcomes between HMAs and observation arms, we focused on comparing the benefits of HMA maintenance following allo-SCTs with an observation approach and further analyzed the efficacy in each HMA subgroup. Notably, OS, RFS, NRM, and CIR were markedly improved with HMA maintenance. Our subgroup analysis demonstrated the advantages of both azacitidine and decitabine use in this setting. In terms of safety, the HMAs were not associated with a higher GVHD incidence. In the case of decitabine for post allo-SCT maintenance, the rate of chronic GVHD seemed to be lower than that of the observation arm. Previous studies showed that HMA maintenance had low rates of toxicities and infectious complications even if the treatment is given to elderly patients (36,37). Furthermore, a prior study reported that 6.8% of post-allo-SCT patients experienced isolated extramedullary relapse which translate into dismal survival outcome (38). Accordingly, prophylaxis scheme post allo-SCT is a rational option to mitigate either isolated extramedullary or bone marrow relapse risk. Taken together, the use of HMAs is a feasible therapy for AML and MDS patients during the post-allo-SCT period, and it should be offered broadly to post allo-SCT patients.
Recently, an oral formulation of azacitidine (CC-486) was approved by the U.S. Food and Drug Administration for the continued treatment of adult AML. Based on data from the phase 3 QUAZAR AML-0001 clinical trial, the patients must have achieved first complete remission or must have an incomplete blood count recovery following intensive induction chemotherapy, and be unable to complete intensive curative therapy (39). The oral formulation of azacitidine may enhance patient convenience, eliminate injection-site reactions, and facilitate long-term administration. The application of this product in a post-allo-SCT setting has since been verified in a phase I/II study, which supports the promising clinical activity (40). A randomized, phase III trial to validate its efficacy is in development. risk index. It is possible that older patients were selected to have less comorbidities, better performance status, and less prior treatment burden compared to the younger ones; which could be the confounding factors behind similar outcomes. Furthermore, the lack of comorbidity is an issue in identifying risk factors for NRM. Third, there is a recent trend in using MRD status in pre-and post-allo-SCT setting to classify MRD-positive patients who would benefit from HMAs maintenance after allo-SCT (41). However, MRD assessment data was scarce in published included trials precluding a subgroup analysis. In addition, the incorporation of other agents, such as gemtuzumab ozogamicin and G-CSF into each treatment protocol also impact outcomes of clinical trials. Largescale randomized trials are warranted to clarify all of these unresolved issues.

CONCLUSION
The current systematic review and meta-analysis illustrated that the patients receiving HMA maintenance post-Allo-SCT had significantly better outcomes with regards to OS, RFS, NRM, and CIR. Furthermore, if decitabine was used for maintenance, the rate of chronic GVHD seemed to be lower than that of the observation arm. Further data, preferably from large prospective studies, is warranted to confirm the benefit of HMA-based maintenance after allo-SCT as well as describe the optimal agent, administration schedule, and the sub-groups of patients who benefit from such intervention.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

ETHICS STATEMENT
The need for ethics approval by institutional board review was waived as this study did not directly involve human subjects.

AUTHOR CONTRIBUTIONS
BP and WO collected the data. BP performed the statistical analyses. WO and SK drafted the manuscript and prepared the final version. FK made critical revisions. All authors designed the study, read, and approved the final manuscript.